This application is for a Clinical Investigator Award to provide support during the completion of my post-doctoral training and subsequent transition into a career as an independent investigator. I received my doctoral training in the NIH-sponsored Medical Scientist Training Program at UCSF, followed by training in Internal Medicine at the Brigham and Women's Hospital, Boston and in Oncology at the Dana-Farber Cancer Institute, Boston. After five years of clinical training, I have returned to the laboratory of Drs. Nicholas Dyson and Ed Harlow in order to study the molecular mechanisms that control cellular proliferation and oncogenesis in humans. cdk3 is a protein kinase that is highly homologous to cdc2 and cdk2, two kinases known to control the cell cycle in animals. All three kinases share the unusual ability to rescue a yeast cell cycle mutation in the cdc2/CDC28 gene. Furthermore, when dominant-negative versions of various cdc2-related kinases were expressed in cells to interrupt endogenous kinase function, only cdc2, cdk2 and cdk3 mutants caused a cell cycle arrest. Interruption of cdk3 activity caused cells to accumulate in G1, indicating that cdk3, like cdk2, may be a key regulator of the G1/S transition. Moreover, cdk3 appears to function independently, since cdk2 cannot substitute for cdk3 in a complementation assay. In mammalian cells, the G1/S transition coincides with a key decision point, where many signals are integrated prior to committing to cell division. Derangements in the signals at this transition are common in cancer cells and a number of its regulators have been revealed as oncogenes or tumor suppressor genes. Compared with cdc2 and cdk2, relatively little is known about cdk3. No cyclin partner has been found, only weak kinase activity has been recorded, and the difference(s) between the roles of cdk2 and cdk3 at the G1/S boundary remain unresolved. Using the yeast two-hybrid system, I have isolated a number of candidate clones for proteins that interact with cdk3. Eleven of these clones are robust in that they demonstrate strong phenotypes in three independent reporter assays. In addition, I have constructed a series of chimeric kinases between cdk2 and cdk3 that enabled me to demonstrate that a sequence in the C-terminal third of cdk3 leads to reduced protein levels. They will also allow me to explore the functional differences between the two kinases in a complementation assay. The studies proposed here are to characterize cdk3 activity by identifying cdk3-associated proteins improving detection of kinase activity, and studying the timing and regulation of that activity in the cell cycle. This work will also explore the differences between cdk2 and cdk3 in their independent roles to govern entry into DNA synthesis. Our laboratory's extensive expertise in the identification and characterization of cell cycle proteins will help me considerably in these experiments.